As cancer continues to be a public health concern, efforts are being directed toward the development of new chemotherapies. Many chemotherapeutic agents induce DNA damage ultimately leading to cell death. Under normal physiological conditions, multiple pathways exist for the repair of damaged DNA. However, such pathways can subvert the effects of chemotherapy targeting DNA in cancer cells and trigger drug resistance. These repair pathways include homologous recombination, non-homologous end joining, nucleotide excision repair, mismatch repair, and base excision repair (BER). While all these pathways repair DNA damage, the types of damage they repair vary, with BER being particularly relevant to exogenous chemical damage such as that caused by chemotherapy.
Damage to DNA can result from direct chemical modification of nucleotides or from accumulation of aberrant bases, such as uracil. Regardless of the type of damage, the first step in the BER pathway is the excision of the damaged base by a glycosylase, which leaves the free ribose sugar termed abasic or AP (apurinic/apyrimidinic) site. For example, uracil DNA glycosylase (UDG) rapidly excises uracil from DNA to initiate the repair sequence (FIG. 3). AP sites are the most common lesions in DNA, and if left unrepaired, can be mutagenic. AP sites are formed following oxidative damage of DNA by reactive oxygen species (ROS) and this oxidative damage is associated with cancer, heart disease, Parkinson disease, and aging. Therefore, tools that detect and quantify AP sites are of broad interest to the medical and scientific communities.
Several research groups have developed tools to detect AP sites based on methods including fluorescence, nanopore ion detection, mass spectrometry, atomic force microscopy, electrochemistry, and electron paramagnetic resonance. Some of these techniques employ a variety of molecular probes targeted to the AP site through various chemical features of the lesions. Several probes containing an aminooxy moiety have been developed that covalently bind to the AP site aldehyde and form an oxime ether. Among them, aldehyde reactive probe (ARP), based on biotin tethered to an alkoxyamine, detects AP sites in a colorimetric streptavidin-horseradish peroxidase in vitro assay.
Previously developed aldehyde-reactive probe (ARP) and similar compounds that fluoresce in the UV-visible range are commonly used for in vitro detection and quantification of AP sites. Such assays have several drawbacks in studying DNA-targeted chemotherapies: 1) they are often limited to the study of AP sites in DNA of circulating cells in plasma induced by chemotherapeutic agents, which is only an indirect measure of AP site formation; 2) for direct detection and quantification of AP sites in tumor regions, dissection and homogenization of tumor tissues are required at each time point after therapeutic treatments, which make longitudinal studies impossible; 3) these assays are not readily repeatable due to sophisticated procedures.